How the dyes affect folding of small proteins in single-molecule FRET experiments: A simulation study

Area of Science:

• Biophysics
• Protein Dynamics
• Biomolecular Simulation

Background:

• Single-molecule Förster resonance energy transfer (smFRET) is crucial for studying protein folding dynamics.
• The influence of labeling dyes on protein behavior, especially for small proteins, remains a critical question.
• Dye size and linker length can be comparable to small protein dimensions, potentially altering conformational landscapes.

Purpose of the Study:

• To investigate the impact of dye labeling on the folding behavior of small proteins.
• To assess how dye-induced excluded volume affects protein conformational space.
• To quantify kinetic differences between unlabeled and labeled protein folding.

Main Methods:

• Coarse-grained molecular dynamics simulations were employed.
• Simulations were performed on the BBL protein and two of its single-molecule Förster resonance energy transfer (smFRET) constructs.
• Analysis focused on conformational changes and folding kinetics.

Main Results:

• The presence of dyes and their excluded volume did not significantly alter the overall folding pathway of the small BBL protein.
• Significant kinetic deviations were observed, with FRET constructs exhibiting slower folding rates compared to the original protein.
• The reduced folding speed in labeled constructs is attributed to the hindrance of initial protein collapse by the massive dyes.

Conclusions:

• Dye labeling has a minimal effect on the fundamental folding mechanism of small proteins like BBL.
• The primary impact of dye labeling on small proteins is a modulation of folding kinetics, specifically a deceleration due to steric hindrance.
• These findings are vital for accurate interpretation of smFRET data in protein folding studies.